Cost-effectiveness of pharmacogenetic testing to predict treatment response to angiotensin-converting enzyme inhibitor

OBJECTIVE: This study aimed to assess the potential cost-effectiveness of testing patients with nephropathies for the I/D polymorphism before starting angiotensin-converting enzyme (ACE) inhibitor therapy, using a 3-year time horizon and a healthcare perspective. METHODS: We used a combination of a decision analysis and Markov modeling technique to evaluate the potential economic value of this pharmacogenetic test by preventing unfavorable treatment in patients with nephropathies. The estimation of the predictive value of the I/D polymorphism is based on a systematic review showing that DD carriers tend to respond well to ACE inhibitors, while II carriers seem not to benefit adequately from this treatment. Data on the ACE inhibitor effectiveness in nephropathy were derived from the REIN (Ramipril Efficacy in Nephropathy) trial. We calculated the number of patients with end-stage renal disease (ESRD) prevented and the differences in the incremental costs and incremental effect expressed as life-years free of ESRD. A probabilistic sensitivity analysis was conducted to determine the robustness of the results. RESULTS: Compared with unselective treatment, testing patients for their ACE genotype could save 12 patients per 1000 from developing ESRD during the 3 years covered by the model. As the mean net cost savings was euro 356,000 per 1000 patient-years, and 9 life-years free of ESRD were gained, selective treatment seems to be dominant. CONCLUSION: The study suggests that genetic testing of the I/D polymorphism in patients with nephropathy before initiating ACE therapy will most likely be cost-effective, even if the risk for II carriers to develop ESRD when treated with ACE inhibitors is only 1.4% higher than for DD carriers. Further studies, however, are required to corroborate the difference in treatment response between ACE genotypes, before genetic testing can be justified in clinical practice.

Role of calcium-independent phospholipase A2 in cortex striatum thalamus cortex circuitry-enzyme inhibition causes vacuous chewing movements in rats

RATIONALE: High levels of calcium independent phospholipase A2 (iPLA2) are present in certain regions of the brain, including the cerebral cortex, striatum, and cerebellum (Ong et al. 2005). OBJECTIVES: The present study was carried out to elucidate a possible role of the enzyme in the motor system. METHODS: The selective iPLA2 inhibitor bromoenol lactone (BEL), the nonselective PLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP), and an antisense oligonucleotide were used to interfere with iPLA2 activity in various components of the motor system. Control animals received injections of carrier (phosphate buffered saline, PBS) at the same locations. The number of vacuous chewing movements (VCM) was counted from 1 to 14 days after injection. RESULTS: Rats that received BEL and high-dose MAFP injections in the striatum, thalamus, and motor cortex, but not the cerebellum, showed significant increase in VCM, compared to those injected with PBS at these locations. BEL-induced VCM were blocked by intramuscular injections of the anticholinergic drug, benztropine. Increased VCM was also observed after intrastriatal injection of antisense oligonucleotide to iPLA2. The latter caused a decrease in striatal iPLA2 levels, confirming a role of decreased enzyme activity in the appearance of VCM. CONCLUSIONS: These results suggest an important role for iPLA2 in the cortex-striatum-thalamus-cortex circuitry. It is postulated that VCM induced by iPLA2 inhibition may be a model of human parkinsonian tremor.

Upregulation of endothelin converting enzyme-1 in host liver during chronic cardiac allograft rejection

Endothelin regulates cytokine expression in vitro and in vivo. This study investigated the effects of chronic allograft rejection on hepatic endothelin-converting enzyme-1 (ECE-1) gene expression and endothelin-1 (ET-1) plasma clearance. Using the Lewis-F344 minor histocompatibility mismatch model of heterotopic cardiac transplantation, hepatic ECE-1 gene expression was measured by real-time polymerase chain reaction and host plasma clearance of ET-1 was measured 8 weeks after transplantation in the absence of immunosuppression. In animals undergoing allograft rejection, hepatic ECE-1 gene expression increased 2-fold (P < 0.05), whereas no effect of rejection on ET-1 clearance from plasma was observed. In summary, upregulation of ECE-1 gene expression occurs in the liver of the host during chronic allograft rejection. Because the liver represents both a key organ for cytokine production and for endothelin metabolism, increased hepatic ECE-1-mediated ET-1 synthesis may contribute to host responses and cytokine production during allograft rejection.

Inhibition of matrix metalloproteinases and tumour necrosis factor alpha converting enzyme as adjuvant therapy in pneumococcal meningitis

Matrix metalloproteinases (MMPs) and tumour necrosis factor alpha (TNF-alpha) converting enzyme (TACE) contribute synergistically to the pathophysiology of bacterial meningitis. TACE proteolytically releases several cell-surface proteins, including the proinflammatory cytokine TNF-alpha and its receptors. TNF-alpha in turn stimulates cells to produce active MMPs, which facilitate leucocyte extravasation and brain oedema by degradation of extracellular matrix components. In the present time-course studies of pneumococcal meningitis in infant rats, MMP-8 and -9 were 100- to 1000-fold transcriptionally upregulated, both in CSF cells and in brain tissue. Concentrations of TNF-alpha and MMP-9 in CSF peaked 12 h after infection and were closely correlated. Treatment with BB-1101 (15 mg/kg subcutaneously, twice daily), a hydroxamic acid-based inhibitor of MMP and TACE, downregulated the CSF concentration of TNF-alpha and decreased the incidences of seizures and mortality. Therapy with BB-1101, together with antibiotics, attenuated neuronal necrosis in the cortex and apoptosis in the hippocampus when given as a pretreatment at the time of infection and also when administration was started 18 h after infection. Functionally, the neuroprotective effect of BB-1101 preserved learning performance of rats assessed 3 weeks after the disease had been cured. Thus, combined inhibition of MMP and TACE offers a novel therapeutic strategy to prevent brain injury and neurological sequelae in bacterial meningitis.

A novel enzyme immunoassay specific for ABCA1 protein quantification in human tissues and cells

ATP-binding cassette transporter A1 (ABCA1) mediates the transport of cholesterol and phospholipids from cells to lipid-poor HDL and maintains cellular lipid homeostasis. Impaired ABCA1 function plays a role in lipid disorders, cardiovascular disease, atherosclerosis, and metabolic disorders. Despite the clinical importance of ABCA1, no method is available for quantifying ABCA1 protein. We developed a sensitive indirect competitive ELISA for measuring ABCA1 protein in human tissues using a commercial ABCA1 peptide and a polyclonal anti-ABCA1 antibody. The ELISA has a detection limit of 8 ng/well (0.08 mg/l) with a working range of 9-1000 ng/well (0.09-10 mg/l). Intra- and interassay coefficient of variations (CVs) were 6.4% and 9.6%, respectively. Good linearity (r = 0.97-0.99) was recorded in serial dilutions of human arterial and placental crude membrane preparations, and fibroblast lysates. The ELISA measurements for ABCA1 quantification in reference arterial tissues corresponded well with immunoblot analysis. The assay performance and clinical utility was evaluated with arterial tissues obtained from 15 controls and 44 patients with atherosclerotic plaques. ABCA1 protein concentrations in tissue lysates were significantly lower in patients (n = 24) as compared with controls (n = 5; 9.37 +/- 0.82 vs. 17.03 +/- 4.25 microg/g tissue; P < 0.01). The novel ELISA enables the quantification of ABCA1 protein in human tissues and confirms previous semiquantitative immunoblot results.

Luminal starch substrate "brake" on maltase-glucoamylase activity is located within the glucoamylase subunit

The detailed mechanistic aspects for the final starch digestion process leading to effective alpha-glucogenesis by the 2 mucosal alpha-glucosidases, human sucrase-isomaltase complex (SI) and human maltase-glucoamylase (MGAM), are poorly understood. This is due to the structural complexity and vast variety of starches and their intermediate digestion products, the poorly understood enzyme-substrate interactions occurring during the digestive process, and the limited knowledge of the structure-function properties of SI and MGAM. Here we analyzed the basic catalytic properties of the N-terminal subunit of MGAM (ntMGAM) on the hydrolysis of glucan substrates and compared it with those of human native MGAM isolated by immunochemical methods. In relation to native MGAM, ntMGAM displayed slower activity against maltose to maltopentose (G5) series glucose oligomers, as well as maltodextrins and alpha-limit dextrins, and failed to show the strong substrate inhibitory "brake" effect caused by maltotriose, maltotetrose, and G5 on the native enzyme. In addition, the inhibitory constant for acarbose was 2 orders of magnitude higher for ntMGAM than for native MGAM, suggesting lower affinity and/or fewer binding configurations of the active site in the recombinant enzyme. The results strongly suggested that the C-terminal subunit of MGAM has a greater catalytic efficiency due to a higher affinity for glucan substrates and larger number of binding configurations to its active site. Our results show for the first time, to our knowledge, that the C-terminal subunit of MGAM is responsible for the MGAM peptide's "glucoamylase" activity and is the location of the substrate inhibitory brake. In contrast, the membrane-bound ntMGAM subunit contains the poorly inhibitable "maltase" activity of the internally duplicated enzyme.

Enzyme engineering of aminotransferases for improved activity and thermostability

The production by biosynthesis of optically active amino acids and amines satisfies the pharmaceutical industry in its demand for chiral building blocks for the synthesis of various pharmaceuticals. Among several enzymatic methods that allow the synthesis of optically active aminoacids and amines, the use of minotransferase is a promising one due to its broad substrate specificity and no requirement for external cofactor regeneration. The synthesis of chiral compounds by aminotransferases can be done either by asymmetric synthesis starting from keto acids or ketones, and by kinetic resolution starting from racemic aminoacids or amines. The asymmetric synthesis of substituted (S)-aminotetralin, an active pharmaceutical ingredient (API), has shown to have two major factors that contribute to increasing the cost of production. These factors are the raw material cost of biocatalyst used to produce it and product loss during biocatalyst separation. To minimize the cost contribution of biocatalyst and to minimize the loss of product, two routes have been chosen in this research: 1. To engineer the aminotransferase biocatalyst to have greater specific activity, and 2. Improve the engineering of the process by immobilization of biocatalyst in calcium alginate and addition of cosolvents. An (S)-aminotransferase (Mutant CNB03-03) was immobilized, not as purified enzyme but as enzyme within spray dried cells, in calcium alginate beads and used to produce substituted (S)-aminotetralin at 50 °C and pH 7 in experiments where the immobilized biocatalyst was recycled. Initial rate of reaction for cycle 1 (6 hr duration) was determined to be 0.258 mM/min, for cycle 2 (20 hr duration) it decreased by ~50% compared to cycle 1, and for cycle 3 (20 hr duration) it decreased by ~90% compared to cycle 1 (immobilized preparation consisted of 50 mg of spray dried cells per gram of calcium alginate). Conversion to product for each cycle decreased as well, from 100% in cycle 1 (About 50 mM), 80% in cycle 2, and 30% after cycle 3. This mutant was determined to be deactivated at elevated temperatures during the reaction cycle and was not stable enough to allow multiple cycles in its immobilized form. A new mutant aminotransferase was isolated by applying error-prone polymerase chain reaction (PCR) on the gene coding for this enzyme and screening/selection: CNB04-01. This mutant showed a significant improvement in thermostability in comparison to CNB03-03. The new mutant was immobilized and tested under similar reaction conditions. Initial rate remained fairly constant (0.2 mM/min) over four cycles (each cycle with a duration of about 20 hours) with the mutant retaining almost 80% of initial rate in the fourth cycle. The final product concentrations after each cycle did not decrease during recycle experiments. Thermostability of CNB04-01 was much improved compared to CNB03-03. Under the same reaction conditions as stated above, the addition of co-solvents was studied in order to increase substituted tetralone solubility. Toluene and sodium dodecylsulfate (SDS) were used. SDS at 0.01% (w/v) allowed four recycles of the immobilized spray dried cells of CNB04-01, always reaching higher product concentration (80-85 mM) than the system with toluene at 3% (v/v) -70 mM-. The long term activity of immobilized CNB04-01 in a system with SDS 0.01% (w/v) at 50 °C, pH 7 was retained for three cycles (20 to 24 hours each one), reaching always final product concentration between 80-85 mM, but dropping precipitously in the fourth cycle to a final product concentration of 50 mM. Although significant improvement of immobilization on productivity and stability were observed using CNB04-01, another observation demonstrated the limitations of an immobilization strategy on reducing process costs. After analyzing the results of this experiment it was seen that a sudden drop occurred on final product concentration after the third recycle. This was due to product accumulation inside the immobilized preparation. In order to improve the economics of the process, research was focused on developing a free enzyme with an even higher activity, thus reducing raw material cost as well as improving biomass separation. A new enzyme was obtained (CNB05-01) using error-prone PCR and screening using as a template the gene derived from the previous improved enzyme. This mutant was determined to have 1.6 times the initial rate of CNB04-01 and had a higher temperature optimum (55°). This new enzyme would allow reducing enzyme loading in the reaction by five-fold compared to CNB03-03, when using it at concentration of one gram of spray dried cells per liter (completing the reaction after 20-24 hours). Also this mutant would allow reducing process time to 7-8 hours when used at a concentration of 5 grams of spray dried cells per liter compared to 24 hours for CNB03-03, assuming that the observations shown before are scalable. It could be possible to improve the economics of the process by either reducing enzyme concentration or reducing process time, since the production cost of the desired product is primarily a function of both enzyme concentration and process time.

Liver enzyme elevation after lamivudine withdrawal in HIV-hepatitis B virus co-infected patients: the Swiss HIV Cohort Study

Background The principal causes of liver enzyme elevation among HIV-hepatitis B virus (HBV) co-infected patients are the hepatotoxic effects of antiretroviral therapy (ART), alcohol abuse, ART-induced immune reconstitution and the exacerbation of chronic HBV infection. Objectives To investigate the incidence and severity of liver enzyme elevation, liver failure and death following lamivudine (3TC) withdrawal in HIV-HBV co-infected patients. Methods Retrospective analysis of the Swiss HIV Cohort Study database to assess the clinical and biological consequences of the discontinuation of 3TC. Variables considered for analysis included liver enzyme, HIV virological and immunological parameters, and medication prescribed during a 6-month period following 3TC withdrawal. Results 3TC was discontinued in 255 patients on 363 occasions. On 147 occasions (109 patients), a follow-up visit within 6 months following 3TC withdrawal was recorded. Among these patients, liver enzyme elevation occurred on 42 occasions (29%), three of them (2%) with severity grade III and five of them (3.4%) with severity grade IV elevations (as defined by the AIDS Clinical Trials Group). Three patients presented with fulminant hepatitis. One death (0.7%) was recorded. Conclusions HBV reactivation leading to liver dysfunction may be an under-reported consequence of 3TC withdrawal in HIV-HBV co-infected patients. Regular monitoring of HBV markers is warranted if active therapy against HBV is discontinued.

Phage lytic enzyme Cpl-1 for antibacterial therapy in experimental pneumococcal meningitis

Treatment of bacterial meningitis caused by Streptococcus pneumoniae is increasingly difficult, because of emerging resistance to antibiotics. Recombinant Cpl-1, a phage lysin specific for S. pneumoniae, was evaluated for antimicrobial therapy in experimental pneumococcal meningitis using infant Wistar rats. A single intracisternal injection (20 mg/kg) of Cpl-1 resulted in a rapid (within 30 min) decrease in pneumococci in cerebrospinal fluid (CSF) by 3 orders of magnitude lasting for 2 h. Intraperitoneal administration of Cpl-1 (200 mg/kg) led to an antibacterial effect in CSF of 2 orders of magnitude for 3 h. Cpl-1 may hold promise as an alternative treatment option in pneumococcal meningitis.

DESIGN AND SYNTHESIS OF MULTIFUNCTIONAL POLYMERIC MICELLES FOR GENE-DIRECTED ENZYME PRODRUG THERAPY

Gene-directed enzyme prodrug therapy is a form of cancer therapy in which delivery of a gene that encodes an enzyme is able to convert a prodrug, a pharmacologically inactive molecule, into a potent cytotoxin. Currently delivery of gene and prodrug is a two-step process. Here, we propose a one-step method using polymer nanocarriers to deliver prodrug, gene and cytotoxic drug simultaneously to malignant cells. Prodrugs acyclovir, ganciclovir and 5-doxifluridine were used to directly to initiate ring-opening polymerization of epsilon-caprolactone, forming a hydrophobic prodrug-tagged poly(epsilon-caprolactone) which was further grafted with hydrophilic polymers (methoxy poly(ethylene glycol), chitosan or polyethylenemine) to form amphiphilic copolymers for micelle formation. Successful synthesis of copolymers and micelle formation was confirmed by standard analytical means. Conversion of prodrugs to their cytotoxic forms was analyzed by both two-step and one-step means i.e. by first delivering gene plasmid into cell line HT29 and then challenging the cells with the prodrug-tagged micelle carriers and secondly by complexing gene plasmid onto micelle nanocarriers and delivery gene and prodrug simultaneously to parental HT29 cells. Anticancer effectiveness of prodrug-tagged micelles was further enhanced by encapsulating chemotherapy drugs doxorubicin or SN-38. Viability of colon cancer cell line HT29 was significantly reduced. Furthermore, in an effort to develop a stealth and targeted carrier, CD47-streptavidin fusion protein was attached onto the micelle surface utilizing biotin-streptavidin affinity. CD47, a marker of self on the red blood cell surface, was used for its antiphagocytic efficacy, results showed that micelles bound with CD47 showed antiphagocytic efficacy when exposed to J774A.1 macrophages. Since CD47 is not only an antiphagocytic ligand but also an integrin associated protein, it was used to target integrin alpha(v)beta(3), which is overexpressed on tumor-activated neovascular endothelial cells. Results showed that CD47-tagged micelles had enhanced uptake when treated to PC3 cells which have high expression of alpha(v)beta(3). The synthesized multifunctional polymeric micelle carriers developed could offer a new platform for an innovative cancer therapy regime.